Fear of disease

The conditioned flight paradigm^{24 (link)} is a Pavlovian fear conditioning paradigm in which an SCS is used as the conditioning stimulus (CS) that is paired with the unconditioned stimulus, a shock. The SCS consists of a 10-s pure tone period (7.5 kHz, 75 dB, 500 ms beeps and 1 Hz) followed by a 10-s white noise period (1–20 kHz, 75 dB, 500 ms bursts and 1 Hz). On the first day (pre-exposure), the SCS alone is presented four times in a white cylinder (27 cm diameter). The conditioning was then performed on two consecutive days, with five SCS/US pairings on each day after a 3-min baseline period (pseudorandomized inter-stimulus interval (ITI): 170–230 s). The conditioning context was a red transparent square box (30 × 30 cm) with a grid floor through which footshocks were delivered. For each SCS/US pairing, a 1-s footshock (0.9 mA; Model 2,100 Isolated Pulse Stimulator, A-M System) immediately followed the last white noise burst. On the fourth day, animals underwent cue retrieval in a transparent cylinder (30 cm diameter), to receive 16 SCS presentations, without US pairings (pseudorandomized ITI: 80–140 s). One group of mice was placed into their HC while replaying the SCS tones instead a new context. A subset of mice underwent an additional day of recording and were placed back in the conditioning context for 15 min on the fifth day of the protocol (context retrieval).
Days 1 and 4 of the conditioned flight paradigm (pre-exposure and cue retrieval) and OF, EPM and LDB recordings were performed in context A (acetic acid smell, dim light), while recordings for days 2, 3 and optionally 5 of the conditioned flight paradigm (conditioning, context retrieval) were made in context E (ethanol smell, brighter light).

All mice were handled for 1 h per day for two weeks prior to the behavioral tests. The behavioral session was conducted in a fear conditioning box (46001, UGO BASILE S.r.l, Italy). Freezing was defined as a complete absence of movement, except for respiration. Scoring of the freezing response duration was started after one second of sustained freezing behavior [34 (link)]. The freezing was measured using the Anymaze (version 6.0, Stoelting) software based on a threshold of change in video image pixels. The mice were delivered with 10 kHz and 1 kHz tones and the percentage of freezing time was averaged for the tone each day.
The changing index of freezing was calculated using the equation: $Thechangingindex=\left(T_{lightonfreezing}-T_{lightofffreezing}\right)/\left(T_{lightonfreezing}+T_{lightofffreezing}\right).$
The discrimination index of fear memory was calculated using the equation: $Thediscriminationindex=\left(T_{10kHzfreezing}-T_{1kHzfreezing}\right)/\left(T_{10kHzfreezing}+T_{1kHzfreezing}\right).$
At the encoding stage (day 1), after 5 min of habituation in context A, the mice received 5 pairings of auditory tone (70 dB SPL 10 kHz or 1 kHz pure tone, 20 s duration) with electric foot shocks (1 mA, 2 s duration, and overlapped with the last 2 s of tone). The time interval between each trial was 70 s. In Fig. 1E, F, and S1C, 10 min before the test, 0.5 µl of pharmacological agent was administered simultaneously bilaterally at a rate of 0.125 µl every 10 s by cannula. The mice were administered either saline, mecamylamine (MCM, 10 µM, 20 µM Aladdin, 826-39-1), or atropine alone (10 µM, 20 µM Aladdin, 5908-99-6). In Fig. 2, each tone is accompanied by optogenetic inhibition or activation. In Fig. 2G, 30 min before the test, mice were intraperitoneally injected with saline and nicotine (10 µM, Sigma Aldrich), and each tone is accompanied by optogenetic inhibition.
At test 0 (day 2), to label tone-responsive ACx neurons, the mice were delivered three times of 10 kHz tone or three times 1 kHz tone.
At test 1a or test 1b (day 2 or day 3), the mice were tested for freezing behavior in response to tests 1a (new fear-unpaired 1 kHz or 10 kHz tone) and 1b (fear-paired 10 kHz or 1 kHz) with a time interval of 2 h in different contexts B and C. The mice were delivered three (Figs. 1, 2, 4, 5 and Figs. S1, 2G, H, 4) or four (Fig. 3, S2E, F) representations of both 10 kHz and 1 kHz tones. In groups with four representations of tones, the first and third tones were accompanied by optogenetic activation or inhibition. In Fig. S2A–C, the mice were only given 10 times optogenetic activations. For test 1, each group of mice was randomly assigned and then equally received 1 kHz or 10 kHz tone in different contexts B and C, and after 90 min, the mice were carefully perfused.
All contexts were different in shape, background, and floor texture.